Thyroid-transcription factor Pax-8 is involved in thyroid-specific expression of thyroglobulin (TG) and thyroperoxidase (TPO) genes. Our previous study demonstrated that DNA-binding activity of Pax-8 is regulated by redox control mechanism. In the present study, we identified redox-sensitive cysteine in Pax-8 molecule. Several mutant Pax-8 cDNAs were constructed by 'enzymatic inverse PCR' to substitute cysteine codon with that of serine. These cDNAs were subjected to in vitro translation, and the binding activities of their products were examined by gel shift assay. Single substitution of Cys45 or Cys57 with serine did not affect the binding of Pax-8. However, when both cysteines were substituted with serines, redox regulation of the DNA binding was abolished, indicating that two cysteine residues, Cys-45 and Cys-57, in the paired domain are responsible for the redox regulation of Pax-8. We next examined redox regulation of thyroid cell functions using antioxidant pyrrolidine dithiocarbamate (PDTC). Rat thyroid FRTL-5 cells were treated with 40 muM PDTC.Northern analysis showed that PDTC induced a marked decrease in TPO and Pax-8 mRNA levels. However, it had no effect on TG and TTF-1 mRNA levels, indicating that PDTC effect is specific to the former two mRNAs. Consistent with the mRNA level, a decrease in DNA-binding activity of Pax-8 was observed. TTF-1 and TTF-2 binding was not altered by PDTC, suggesting that TPO expression is mainly regulated by Pax-8. Treatment of FRTL-5 cells with PDTC also induced a remarkable increase in thymidine incorporation, indicating increased cell growth. These results suggest that redox regulation is involved in the expression of differentiation markers and proliferation of thyroid cells.